Higher-Order Chromatin Organization Using 3D DNA Fluorescent In Situ Hybridization.

Abstract

A comprehensive analysis of the tridimensional (3D) organization of the genome is crucial to understand gene regulation. Three-dimensional DNA fluorescent in situ hybridization (3D-FISH) is a method of choice to study nuclear organization at the single-cell level. The labeling of DNA loci of interest provides information on their spatial arrangement, such as their location within the nucleus or their relative positioning. The single-cell information of spatial positioning of genomic loci can thus be integrated with functional genomic and epigenomic features, such as gene activity, epigenetic states, or cell population averaged chromatin interaction profiles obtained using chromosome conformation capture methods. Moreover, the development of a diversity of super-resolution (SR) microscopy techniques now allows the study of structural chromatin properties at subdiffraction resolution, making a finer characterization of shapes and volumes possible, as well as allowing the analysis of quantitative intermingling of genomic regions of interest. Here, we present and describe a 3D-FISH protocol adapted for both conventional and SR microscopy such as 3D structured illumination microscopy (3D-SIM), which can be used for the measurement of 3D distances between loci and the analysis of higher-order chromatin structures in cultured Drosophila and mammalian cells.